In an effort to further extend the number of targets for development of anti-retroviral agents, we are studying HIV-1 integrase inhibitors using in vitro assays using recombinant enzyme and short oligonucleotides corresponding to the proviral ends (LTR's). Integrase is a rationale target for inhibitor development because it is essential for viral replication. It is encoded by the viral genome and does not have a cellular equivalent. Our laboratory has pioneered this research field and reported various families of inhibitors. We have written several reviews on this topic; the most recent will be published in early 2004. This year, the first class of HIV-1 integrase inhibitors has been introduced in clinical trials. We are investigating these diketo acid (DKA) derivatives in collaboration with Dr. Terrence Burke (Laboratory of Medicinal Chemistry, CCR, NCI) and Dr. Vinay Pathak (Antiviral Drug Resistance Program, CCR, NCI). We have elucidated the structure-activity relationship for this new type of compounds, and found that DKAs discriminate between wild-type and mutant HIV-1 integrase and between magnesium and manganese, the two metal cofactors for integrase. Our goal is to elucidate the drug binding site(s) in the enzyme-DNA complex, and to discover agents with a greater therapeutic index and/or novel structural motifs. We discovered that azido derivatives of diketo acids are potent and selective anti-integrase inhibitors, and are antiviral. The azido group can chelate the divalent metal in the enzyme catalytic site. A patent application has been filed for these derivatives. We are also studying novel types of inhibitors that can prevent integration by binding to the proviral DNA ends as well as small peptide and nucleotide inhibitors. The peptide inhibitors are also antiviral and have been patented. We are also studying the molecular interactions between integrase and its DNA substrate using enzyme-DNA crosslinking assays in order to model drug-enzyme-DNA interactions. A study using minor groove scanning with benzo[a]pyrene diol epoxide dG-N2 adduct is in press in The Journal of Biological Chemistry. We recently found a novel interaction between one of the viral DNA bases and the enzyme.